Project Summary Pro-inflammatory cytokine TNF? is believed to be responsible for the delayed fracture healing observed in diabetes. However, there is no consensus on the effect of TNF? inhibition on the bone formation, indicating the unmet need in searching for new regents with unique features other than pure TNF inhibitors for diabetic fracture healing. Our genetic screen led to the identification of TNFR as the novel receptor of progranulin (PGRN) (Tang, et al, Science, 2011), a chondrogenic factor that has been shown to be therapeutic against autoimmune inflammatory arthritis. Type 1 diabetes is the most common autoimmune disease, characterized by chronic inflammation and elevated TNF? activity. Although TNF? activity is mediated primarily through TNFR1, we were excited to find that PGRN-stimulated bone regeneration largely depends on TNFR2. These paradoxical findings suggest that the regenerative PGRN/TNFR2 pathway plays a major role in PGRN-stimulated fracture healing. In addition, 14-3-3? was identified as a component of TNFR2 pathway in response to PGRN stimulation. Further, we have developed an engineered protein named Atsttrin which is composed of three TNFR-binding domains of PGRN, and Atsttrin is more effective than PGRN in inflammatory arthritis. Given that elevated TNF? is believed to be responsible for delayed diabetic fracture healing, we hypothesize that PGRN and Atsttrin stimulate diabetic fracture healing through a) inhibition of TNF?/TNFR1 inflammatory and bone resorption pathway; and primarily b) recruitment of 14-3-3? to TNFR2, followed by activation of bone regeneration pathway. The Specific Aims are: (1) To determine the role of PGRN, especially its derivative Atsttrin, in diabetic fracture healing. We will use both systemic and inducible PGRN knockout mice to determine whether knockout of PGRN delays diabetic fracture healing, and whether recombinant PGRN and Atsttrin can reverse it (SA#1A); which stage of fracture healing requires PGRN for successful completion of diabetic fracture healing (SA#1B); and whether PGRN, especially Atsttrin, has therapeutic efficacy in treating diabetic fracture (SA#1C). We will use an appropriate injectable hydrogel to locally deliver various dosages of PGRN or Atsttrin. (2) To elucidate the molecular mechanisms by which PGRN and Atsttrin stimulate diabetic fracture healing. We will determine the effects of PGRN, Atsttrin, and TNF? on chondrogenesis of diabetic bone marrow stem cells, signaling pathways, interplays and dependence on TNFR and 14-3-3? (SA#2A); whether both TNFRs are important for mediating PGRN's role in diabetic bone healing (SA#2B); and whether the protective effects of PGRN and Atsttrin depend on 14-3-3? by establishing diabetic fracture models with inducible 14-3-3?[-/-] mice (SA#2C). Proposed studies will not only advance our understanding of the molecular events underlying diabetic fracture healing, but could also lead to novel therapeutic interventions for diabetic fracture healing and other conditions in which fracture healing is impaired.